Infection of a host cell by an enveloped virus is initiated by binding of at least one viral envelope protein to a cognate cellular virus receptor protein on the cell surface. The viral envelope protein binds to the receptor and mediates fusion of the viral envelope and the host cell membrane. The presence or absence on a cell of a cognate cellular virus receptor protein is a primary determinant of the host range and the tissue tropism of a virus.
Although an enveloped virus preferentially incorporates its own viral envelope protein(s) into the envelope during virus assembly, the tropism of a number of enveloped viruses may be altered when a different viral envelope glycoprotein is incorporated into the envelope during virus assembly by a process called phenotypic mixing or pseudotyping. Virus pseudotypes may be formed by co-infection of a cell by two different enveloped viruses or may be generated experimentally by expressing a viral envelope protein encoded by one virus in a cell infected with another virus. Pseudotype formation in vivo has been postulated to enhance or alter the pathologic potential of an enveloped virus.
In addition to other viral envelope proteins, enveloped viruses may also incorporate a number of host surface proteins, including cellular virus receptor proteins, into their envelopes (Bubbers et al., 1977, Nature 266:458-459; Lodish et al., 1980, Cell 19:161-169; Calafat et al., 1983, J. Gen. Virol. 64:1241-1253). For example, class I and class II major histocompatibility complex proteins, ICAM-1, ICAM-2, ICAM-3, CR3, CR4, CD43, CD44, CD55, CD59, CD63 and CD71 have been identified in the viral envelope of human immunodeficiency virus, HIV-1 (Bastiani et al., 1997, J. Virol. 71:3444-3450). Similarly, CD55 and CD59 have been identified in human cytomegalovirus virions and also in human T cell leukemia virus virions (Spear et al., 1995, J. Immunol. 155:4376-4381). The measles cellular virus receptor, CD46, has been reported in the envelope of HIV-1 (Montefiori et al., 1994, Virol. 205:82-92). In addition, transient high level expression in cultured cells of CD4, the primary cellular receptor for HIV-1, causes CD4 to partition into the envelope of a number of viruses, including retroviruses, herpesviruses, and rhabdoviruses (Dolter et al., 1993, J. Viral. 67:189-195; Schnell et al., 1996, Proc. Natl. Acad. Sci. USA 93:11359-11365; Schubert et al., 1992, J. Viral. 66:1579-1589; Young et al., 1990, Science 250:1421-1423). A number of factors influence the efficiency of cellular protein uptake by enveloped viruses including the surface density of the cellular protein, the location of the cellular protein within a cellular membrane, and the structural configuration of the cellular protein (Young et al., 1990, Science 250:1421-1423; Suomalainen et al., 1994, J. Virol. 68:4879-4889). Although there have been several reports of incorporating cellular virus receptor proteins into viruses, the structural and functional integrity of these proteins was not suggested, tested, used, or demonstrated previously (Montefiori et al., 1994, Virol. 205:82-92; Dolter et al., 1993, J. Virol. 67:189-195; Schnell et al., 1996, Proc. Natl. Acad. Sci. USA 93:11359-11365; Schubert et al., 1992, J. Virol. 66:1579-1589; Young et al., 1990, Science 250:1421-1423).
Intracellular immunization as a method of gene therapy has been proposed as a potential treatment for AIDS (Friedman et al., 1988, Science 335:452-454; Baltimore, 1988, Science 335:395-396). It has been proposed that the immune system of an AIDS patient may be reconstituted with hematopoietic stem cells that have been rendered resistant to viral infection by the introduction of a gene or a plurality of genes which protect the cell against HIV infection. Numerous intracellular antagonists of HIV replication have been developed which exhibit potent antiviral properties in vitro including trans-dominant mutants (Bevec et al., 1992, Proc. Natl. Acad. Sci. USA 89:9870-9874; Malim et al., 1992, J. Exp. Med. 176:1197-1201; Bahner. et al., 1993, J. Virol. 67:3199-3207; Nabel et al., 1994, Hum. Gene Ther. 5:79-92), molecular decoys (Sullenger et al., 1990, Cell 63:601-608; Lee et al., 1992, New Biol. 4:66-74; Lee et al., 1994, J. Virol. 68:8254-8264), intracellular antibodies (Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893; Duan et al., 1994, Proc. Natl. Acad. Sci. USA 91:5075-5079; Shaheen et al., 1996, J. Virol. 70:3392-3400; Levy-Mintz et al., 1996, J. Virol. 70:8821-8832), anti-sense RNA (Lo et al., 1992, Virology 190:176-183; Sczakiel et al., 1992, J. Viral. 66:5576-5581; Biasolo et al., 1996, J. Virol. 70:2154-2161), ribozymes (Sarver et al., 1990, Science 247:1222-1225; Yamada et al., 1994, Gene Therapy 1:38-45; Heusch et al., 1996, Virology 216:241-244), and other antagonists (Caruso et al., 1992, Proc. Natl. Acad. Sci. USA 89:182-186; Curiel et al., 1993, Hum. Gene Ther. 4:741-747; Brady et al., 1994, Proc. Natl. Acad. Sci. USA 91:365-369).
CD4 has long been known as the cell-surface protein necessary for HIV binding to and entry into cells (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-771; Maddon et al., 1986, Cell 47:333-348). Hence, CD4 is a cellular virus receptor protein for HIV. Recently, chemokine receptors have been shown to also be components of the HIV receptor complex and are important determinants of HIV-1 tropism (Bates, 1996, Cell 86:1-3; D'Souza et al., 1996, Nature Med. 2:1293-1300; Weiss, 1996, Science 272:1885-1886). HIV envelope proteins interact with CD4 and chemokine receptors present on the surface of a cell, resulting in binding of the virus to the cell followed by virus-mediated fusion of the viral envelope and the cell membrane. Initial binding of the HIV envelope protein, gp120, to CD4 triggers one or more conformational changes in one or more HIV viral envelope glycoproteins. Subsequent interaction of an HIV envelope protein with a chemokine receptor on the surface of the cell causes exposure of the viral gp41 fusion peptide to the cell membrane, and ultimately results in fusion of the viral envelope and cell membrane (Collman et al., 1989, J. Exp. Med. 170:1149-1163).
Chemokine receptors are seven transmembrane-spanning G-protein coupled receptors and are divided into two classes, the CC-class and the CXC-class of chemokine receptors. These two classes of chemokine receptors differ in their tissue distribution, their ligand specificity, and their capacity to specifically interact with particular viruses, including particular isolates of HIV and SIV. CCR5 is a chemokine receptor in the CC-class and CXCR4 is a chemokine receptor in the CXC-class.
The ability of HIV-1 to infect T-cells is well known. T-cell tropic strains of HIV-1 undergo envelope-mediated fusion with and enter into T-cells only if the T-cells express both CXCR4 and CD4 (Berson et al, 1996, J. Virol. 70:6288-6295; Feng et al., 1996, Science 272:872-877).
Macrophages and other mononuclear phagocytes are an important reservoir for virus replication in HIV-infected individuals and are suspected to be a major source of ongoing virus replication in patients receiving anti-retroviral therapy (Gartner et al., 1986, Science 233:215-219; Ho et al., 1995, Nature 373:123-126; Wei et al., 1995, Nature 373:117-122; Coffin, 1995, Science 267:483-489). Macrophage-tropic strains of HIV-1 undergo envelope mediated fusion with and enter into macrophages only if the macrophages express both CCR5 and CD4 (Choe et al., 1996, Cell 85:1135-1148; Doranz et al., 1996, Cell 85:1149-1158; Deng et al., 1996, Nature 381:661-666; Dragic et al., 1996, Nature 381:667-673; Alkhatib et al., 1996, Science 272:1955-1958). Similarly, SIV undergoes envelope mediated fusion with and enters into cells only if the cells express both CCR5 and CD4, although other unidentified cellular virus receptor proteins have been also implicated in SIV infection (Chen et al., 1997, J. Virol. 71:2705-2714; Edinger et al., 1997, Proc. Natl. Acad. Sci. USA 94:4005-4010; Marcon et al., 1997, J. Viral. 71:2522-2527). Many primary isolates of HIV-1 are capable of undergoing envelope mediated fusion with and entering into cells which express CD4 and at least one chemokine receptor, including, but not limited to, CXCR4 and CCR5 (Choe et al., 1996, Cell 85:1135-1148; Doranz et al., 1996, Cell 85:1149-1158; Simmons et al., 1996, J. Virol. 70:8355-8360; Connor et al., 1997, J. Exp. Med. 185:621-628; He et al., 1997, Nature 385:645-649). Therefore, expression of chemokine receptors or other cellular virus receptor proteins on the surface of cells appears to be a major determinant of enveloped viral tropism.
Young et al. have demonstrated that CD4 is efficiently incorporated into the envelopes of retroviral particles. However, these particles failed to enter cells expressing HIV envelope glycoproteins (Young et al., 1990, Science 250:1421-1423). Recently, Schnell et al. reported that CD4 may be packaged into vesicular stomatitis virus (Schnell et al., 1996, Proc. Natl. Acad. Sci. USA 93:11359-11365). Thus, to date, production of virus particles comprising host cell receptors or other surface proteins while preserving the biological function of the molecule, has not been achieved.
Ligand interactions with membrane proteins are responsible for a multitude of cell adhesion, signaling, and regulatory events. This diversity of functions makes membrane proteins, such as seven transmembrane domain (7TM) receptors, important drug targets. Proteins that span the membrane multiple times present a unique set of challenges for ligand binding studies because they require a lipid environment to maintain native structure. Whereas detergent conditions can occasionally be found that allow native structure to be maintained in solution, this is an empirical and frequently time-consuming process. As a result, ligand binding studies involving 7TM and many other membrane proteins typically involve using whole cells or vesicles derived from cell membranes, where the protein of interest is a minor component.
Interactions between the HIV-1 envelope (Env) protein and its receptors underscore both the strengths and weaknesses of cell-surface binding assays. HIV-1 Env mediates virus entry by sequentially binding to CD4 and a coreceptor, with these interactions triggering conformational changes in Env that lead to membrane fusion (Berger et al., 1999, Annu. Rev. Immunol. 17:657-700). R5 virus strains that are responsible for virus transmission use the 7TM chemokine receptor CCR5 in conjunction with CD4 to enter cells, X4 virus strains that tend to evolve years after infection use the related CXCR4 receptor, and intermediate dual-tropic R5X4 virus strains can use both receptors. Binding of the soluble gp120 subunit of Env to CD4 is readily detected, and gp120 proteins from some R5 virus strains bind to CCR5 with high affinity (Doranz et al., 1999, J. Virol. 73:10346-10358 and Doranz et al., 1999, J. Viral. 73:10346-40358). However, direct binding of X4 gp120 proteins to CXCR4 has been difficult to measure, as has binding of R5X4 gp120 proteins to either CXCR4 or CCR5 (Doranz et al., 1999, J. Virol. 73:2752-2761, Baik et al., 1999, Virology 259:267-273 and Etemad-Moghadam et al., 2000, J. Viral. 74:4433-4440). Interactions between Env and alternative coreceptors such as CCR3 and STRL33 also cannot be measured using standard binding techniques (Baik et al., 1999, Virology 259:267-273). As virus receptor interactions can be the targets of neutralizing antibodies and small molecule inhibitors (reviewed in ref. 1), improved assays to measure these binding events are needed.
An approach that in principle would make it possible to monitor low affinity but functionally important Env-coreceptor interactions would be to use microfluidic devices, e.g., biosensors (optical and SPR biosensors), and other analytical instruments that detect interactions between molecules, preferably in real-time. The most commonly used optical biosensors (Biacore, Uppsala, Sweden) are based on surface plasmon resonance, which measures changes in refractive index at the sensor surface (Canziani et al., 1999, Methods 19:253-269 and Rich et al., 2000, Curr. Opin. Biotechnol. 11:54-61). With this technique, one protein is tethered to the biosensor surface, and changes in refractive index that occur upon exposure to its binding partner are monitored. However, a general method for attaching intact membrane proteins to this instrument does not exist. Membrane proteins can span the membrane multiple times, can form homo- or hetero-oligomers in the membrane, and removal from the lipid bilayer can destroy tertiary or quaternary structure. Thus, despite the importance of membrane proteins in biological processes, to date, there is no method to study the complex interaction between these molecules and molecules that specifically interact with them using powerful techniques such as, but not limited to, using optical biosensors.
To date, there are a limited number of therapies directed against HIV infection in humans, each having a variable success rate, generally concomitant with the emergence of strains of HIV which are resistant to the therapy. There remains an acute need for the development of anti-HIV therapies to which the virus cannot develop resistance. The present invention satisfies this need.
Further, there is long-felt need for assays for the study of cell membrane protein-protein interactions, and the present invention also satisfies this need.